Number Of Non-viable Cells Research Articles

Inactivation of Salmonella in ready-to-eat cabbage slices packaged in a plastic container using an integrated in-package treatment of hydrogen peroxide and cold plasma

An in-package cold plasma (CP) treatment integrated with hydrogen peroxide (H 2 O 2 ) treatment was developed for the inactivation of foodborne pathogens in cabbage slices packaged in polyethylene terephthalate (PET) containers. The effects of H 2 O 2 -CP treatment on microbial inactivation, microbial cell membrane integrity, cell oxidation, and the antioxidant activity and color of cabbage slices were investigated. As the treatment time increased from 1 to 2 and 3 min, the respective reduction level of Salmonella increased from 0.6 to 0.9 log CFU/g ( p < 0.05). Moreover, the levels of Salmonella reduction were 0.8, 0.9, 1.8, and 1.5 log CFU/g when the H 2 O 2 concentrations in the H 2 O 2 pad within the package were 3, 10, 20, and 30%, respectively. The values for cell lipid peroxidation, reactive oxygen species generation, and number of non-viable cells with damaged membranes, increased following CP and H 2 O 2 -CP treatments. Meanwhile, H 2 O 2 -CP treatment did not influence the antioxidant activities nor the color of cabbage slices. However, the concentration of malondialdehyde in cabbage slices was increased by H 2 O 2 -CP treatment. The results suggest that H 2 O 2 -CP treatment is an effective strategy for disinfecting cabbage slices without altering their antioxidant activities and color. • In-package cold plasma (CP) and H 2 O 2 treatment inactivates Salmonella effectively. • Increased treatment time enhances reduction of Salmonella in cabbage slices. • H 2 O 2 decreases the number of Salmonella in a concentration dependent manner. • CP/H 2 O 2 -CP treatments increase lipid peroxidation, ROS, and non-viable cell number. • H 2 O 2 -CP treatment does not impact antioxidant activities or color of cabbage slices.

Polylactide-based stent coatings: biodegradable polymeric coatings capable of maintaining sustained release of the thrombolytic enzyme streptokinase

Abstract The paper describes synthesis and testing of novel biodegradable polylactide-based polymer membranes with desired mechanical properties, which are capable of sustained and directed release of biomacromolecules with high molecular weight (in particular, streptokinase; m.w. 47 kDa). Streptokinase is a pharmaceutical agent, possessing a pronounced thrombolytic activity. The membranes synthesized had a percentage elongation of 2–11% and tensile strength of 25–85 MPa. They were biodegradable – yet being stored in aqueous media in the absence of biological objects, would be dissolved by no more than 10% in 6 months. The synthesized membranes were capable of controlled release of streptokinase into the intercellular space, with the enzyme retaining more than 90% of its initial activity. The rate of streptokinase release from the membranes varied from 0.01 to 0.04 mg/day per cm2 of membrane surface. The membrane samples tested in the work did not have any short-term toxic effects on the cells growing de novo on the membrane surface. The mitotic index of those cells was approximately 1.5%, and the number of non-viable cells on the surface of the polymer films did not exceed 3–4% of their total amount. The implantation of the synthesized polymers – as both individual films and coatings of nitinol stents – was not accompanied by any postoperative complications. The subsequent histological examination revealed no abnormalities. Two months after the implantation of polymer films, only traces of polylactide were found in the implant-surrounding tissues. The implantation of stents coated with streptokinase-containing polymers resulted in the formation of a mature and thick connective-tissue capsules. Thus, the polylactide membranes synthesized and tested in this work are biodegradable, possess the necessary mechanical properties and are capable of sustained and directed release of streptokinase macromolecules.

Antimicrobial mechanism of luteolin against Staphylococcus aureus and Listeria monocytogenes and its antibiofilm properties

Luteolin (LUT) is a naturally occurring compound found in a various of plants. Few recent studies have reported LUT antimicrobial activities against bacterial pathogens, however, the fundamental LUT mediated antimicrobial mechanism has never been elucidated. This study aimed to investigate the antimicrobial activities of LUT and its mode of action against Staphylococcus aureus and Listeria monocytogenes, either as planktonic cells or as biofilms. Here, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of LUT against S. aureus and L. monocytogenes were determined using the broth microdilution method, and the antimicrobial mode of LUT was elucidated by evaluating the variations in both cell membrane integrity and cell morphology. Moreover, the biofilm inhibition was measured by crystal violet staining assay, while its qualitative imaging was achieved by confocal laser scanning microscope and field emission scanning electron microscope. MIC and MBC values of LUT against S. aureus were 16–32 and 32–64 μg/mL, and 32–64 and 64–128 μg/mL for L. monocytogenes. LUT destroyed the cell membrane integrity, as evidenced by a significant increase in the number of non-viable cells, and well-defined variations in cell morphology. Moreover, LUT presented robust inhibitory effects on the biofilm formation, enhanced antibiotics diffusion within biofilms and killed efficiently mono- and dual-species biofilm cells. Overall, LUT demonstrates potent antimicrobial properties on planktonic and biofilm cells, and the biofilm formation, and thus has the potential use as a natural food preservative in foods.

In vitro Susceptibility and Evaluation of Techniques for Understanding the Mode of Action of a Promising Non-antibiotic Citrus Fruit Extract Against Several Pathogens.

The screening for alternatives to antibiotics is an urgent need for the pharmaceutical industry. One of these alternatives seems to be the citrus fruit extracts, which are showing a significant antibacterial activity against Gram-negative and Gram-positive bacteria. One of these citrus extracts, named BIOCITRO®, is assessed in this study to elucidate its bacteriostatic and bactericidal effect and its mode of action on the important pathogens Campylobacter coli, C. jejuni, Escherichia coli, Salmonella enterica ssp. enterica, Clostridium difficile, C. perfringens, and Staphylococcus aureus. For most of the strains tested of these bacteria the product was bactericidal as well as bacteriostatic at the same concentration, and the minimum bactericidal concentrations ranged from 16 to 256 μg/mL. Regarding the mode of action, important changes in the permeability, structure, composition and morphology of the bacterial envelope were evidenced using flow cytometry, Fourier transform infrared spectroscopy and scanning electron microscopy. The main effect of the product was found over carbohydrates and polysaccharides, inducing the release of microvesicles by the cells in addition to other specific effects.During the study, the techniques used were evaluated to clarify their contribution to the knowledge of the mode of action of the product. The survival test elucidated whether the modifications displayed using other techniques affected the viability of the cells or on the contrary, the cells remained viable even with evident changes in their structure, composition or morphology. Flow cytometry showed that for some strains the proportion of cells detected with altered membrane permeability were higher than the number of non-viable cells, and therefore the damage did not affect the viability of some cells. On the contrary, some cells observed using scanning electron microscopy with no apparent damage, were demonstrated non-viable using the survival test, making this technique indispensable in studies of the mode of action of antimicrobials to make a correct interpretation of the data from other techniques.

49 Collection of Day 7 equine embryos in aluteal cycles in mares

Serial administration of prostaglandin F2α (PGF2) in early diestrus has been recently shown to prevent luteal function in mares and is associated with delayed embryo development in induced aluteal cycles (progesterone deprived environment; &amp;lt;1.0ng mL−1; Leisinger et al. 2018 Theriogenology 105, 178-183). We hypothesised that embryos collected on Day 7 during induced aluteal cycles would be developmentally delayed compared with those collected from control cycles (progesterone deprived environment but supplemented with exogenous progestogen). Mares were monitored until a preovulatory follicle=35mm and the presence of uterine oedema were detected by ultrasonography. Mares were treated IV once with 2000IU of human chorionic gonadotropin and artificially inseminated every other day with total motile spermatozoa from one stallion of known fertility until ovulation. Mares were examined twice daily to determine the occurrence of ovulation. After ovulation, mares were randomly assigned to the control group (serial PGF2 treatment+long-acting altrenogest) or AL group (serial PGF2 treatment only). Using a protocol to induce aluteal cycles (Leisinger et al. 2018 Theriogenology 105, 178-183), mares in the AL group (n=7) were treated twice daily with 10mg of PGF2 (Lutalyse, dinoprost tromethamine, Zoetis, Florham Park, NJ, USA) IM on Days 0, 1, 2, and then once daily on Days 3 and 4. Mares in the control group (n=4) were treated with serial PGF2 treatment as mentioned before, and treated with a single injection of 225mg altrenogest (BioRelease Altrenogest LA 150; BET Pharm, Lexington, KY, USA) IM at the time of ovulation. On Day 7 post-ovulation, embryo collection was performed by uterine flushing using lactated Ringer’s solution. The developmental stage of embryos, diameter, and quality were determined using a stereomicroscope and photographed. Embryos were washed 3 times in commercial embryo holding medium (EmCare™ Holding Solution, ICP Bio, Spring Valley, WI) and stained with 1µg mL−1 of 4’,6-diamidino-2-phenylindole at 38.5°C for 15min to determine the number of nonviable cells. Data were analysed using t-test or Mann-Whitney U test where appropriate. Statistical significance was set at P=0.05. Data are reported as mean±s.e.M. Overall, the developmental stage of control embryos differed from AL embryos (P&amp;lt;0.03). In the control group, all embryos (n=4) collected were classified as expanded blastocysts. In contrast, embryos (n=7) collected from the AL group were all early blastocysts. Furthermore, the mean embryo diameter differed in control v. AL groups, 401±8mm v. 166±2mm, respectively (P&amp;lt;0.001). The quality grades of control embryos (1.3±0.3) were not different from the AL group (1.3±0.2; P&amp;gt;0.05). All embryos of both groups had less than 10% of blastomeres stained with 4’,6-diamidino-2-phenylindole. This study showed that morphologically normal equine embryos of acceptable quality can be collected on Day 7 in aluteal cycles.

Biocompatibility of the Ti81Nb13Ta3Zr3 Alloy

Titanium-based alloy with low-toxic metals such as niobium, tantalum and zirconium (TiNbTaZr) was manufactured. The primary weight percentages of elements used for fusion were: titanium, 65%; niobium, 20%, tantalum, 10%; and zirconium, 5%. The TiNbTaZr alloy had a yield strength of about 550 MPa, a tensile strength of about 700 MPa, and a Young’s modulus of about 50 GPa, that is, it was comparable with Nitinol. A primary study of the biocompatibility of the TiNbTaZr alloy using the SH-SY5Y cells showed that the alloy did not have significant short-term toxicicity towards the cells incubated on the alloy surface. The number of non-viable cells was 2.5 times lower on the TiNbTaZr alloy than on Nitinol. The biocompatibility of TiNbTaZr was more pronounced than that of the Nitinol reference sample.

Ethyl Acetate Extract of &amp;lt;i&amp;gt;Senna alata&amp;lt;/i&amp;gt; (L) Roxb Increases Cytotoxicity in the Human Breast, Prostate and Colorectal Cancer Cells

The objective of this study is to evaluate the effects of ethyl acetate extract of Senna alata (L) Roxb on some human carcinomas - MCF 7 (human breast), C4-2WT (prostate), HT 29 and HTC 116 (colorectal) cell lines. Screening assays carried out to determine cytotoxicity include: - MTT (3- (4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), clonogenic cell survival, Trypan Blue exclusion and methylene blue assays. Evaluation of the results showed that the extract strongly decreased the proliferation of the carcinoma cells in a dose-dependent manner. The minimum concentration of the extract required for 50% inhibition (GI50) of the different cell lines calculated after MTT test were as follows: MCF-7 = 5.90 µg/ml, HCT 29 = 4.97 µg/ml, HCT 116 =11.86 µg/ml and C4-2WT = 9.48 µg/ml. Trypan Blue exclusion assay showed a decrease in the number of viable cells and an increase in the number of non-viable cells over 72 hrs post-treatment with the extract. Methylene blue assay showed that the number of viable cells, when their optical densities were measured over 72 hrs post-treatment, was reduced compared with the control. For clonogenic cell survival, there was an increase in cell proliferation and colony formation in the control cultures. However, cells treated with GI50 and 2X (twice) GI50 concentration of the extract showed a decrease in the number of colonies formed. The results indicate the cytotoxic potentials of the extract and therefore, suggests the use of ethyl acetate leaf extract of Senna alata (L) Roxb in preparing recipes for the management of cancer-related ailments.

Development and In Vitro Assessment of Nanoemulsion for Delivery of Ketoconazole Against Candida albicans

Nanoemulsions (NEs) applied as drug delivery systems are particularly convenient to increase solubility and the release of the drug with poor water solubility in water. The aim of this study was to prepare NEs containing Ketoconazole (KTZ), a poorly water-soluble drug, characterize them and evaluate their antifungal activity against Candida albicans. The solubility of KTZ was studied in different essential oils. Clove and sweet fennel essential oils were chosen as the oily phase and Pluronic F127® and Cremophor RH40® as the non-ionic surfactants to take part in the water phase. Formulations were prepared by a high-energy emulsification method and they were evaluated in terms of physical appearance, mean droplet size and polydispersity index. The NEs were successfully obtained and the droplets mean size was less than 100 nm. To verify efficacy of the NEs, the viability and the growth of C. albicans were measured by the propidium iodide influx analysis and by counting colony-forming units (CFU/mL). The results showed that the loss of the fungal membrane integrity and growth decrease were obtained after the treatment with the KTZ NEs containing clove oil (NEs-CL-KTZ) and with the free KTZ. However, the number of non-viable cells were significantly (p &lt; 0.01) higher on the cells treated with NEs-CL-KTZ compared to the free KTZ. The In Vitro drug release profile demonstrated that NEs-CL-KTZ formulation could increase more than nine times the release of KTZ when compared to KTZ cream. To conclude, NEs-CL-KTZ tested in the present study has demonstrated the most efficient formulation for the treatment of C. albicans infections.

In vitro and in vivo anticancer properties of cucurbitacin isolated from Cayaponia racemosa

Context: Cucurbitacins are a group of triterpenoids that have a cucurbitane skeleton with a wide range of biological activities.Objectives: This study evaluated the anticancer properties of one cucurbitacin isolated from Cayaponia racemosa Cong. (Cucurbitaceae), 2β,3β,16α,20(R),25-pentahydroxy-22-oxocucurbita-5-en (1), with in vitro and in vivo models.Materials and methods: In vitro cytotoxic activity was determined with human leukemia (HL60) and normal blood cells (PBMC). Sarcoma 180 was used as in vivo model.Results: The cucurbitacin (1) reduced the number of viable cells; however, there was no changed in the number of non-viable cells at 5 µg/mL. Selectivity towards cancer cells was suggested by the absence of activity on normal proliferating lymphocytes at the concentrations tested (IC50 >25 µg/ml). Morphological analysis of compound 1-treated cells showed typical apoptotic features, such as intense deposition of granules in the cytoplasm (eosinophilia), DNA fragmentation and irregularities in the plasma membrane. In addition, the cells treated with compound 1 presented intense vacuolization and disruption of the plasma membrane. Acridine orange/Ethidium bromide staining confirmed these findings, revealing an increased number of apoptotic cells. In the Sarcoma 180 tumor model, compound 1 showed 52 and 62% of antitumor activity, either alone (25 mg/kg/day) or in association with the chemotherapeutic agent 5-FU (10 + 10 mg/kg/day), respectively. Moreover, either alone or associated with 5-FU, treatment with compound 1 caused an increase in spleen weight and morphological alterations related to immunostimulatory properties.Conclusion: These data indicate that these naturally occurring compounds have anticancer potential.

Role of Antibiofilm-Antimicrobial Agents in Controlling Device-Related Infections

To assess the effects of N-acetylcysteine (NAC) on organism viability in planktonic and biofilm phases, biofilm thickness, and extracellular polysaccharide content. We performed time-kill curves and broth macrodilution assays of bacterial and fungal clinical isolates with varying concentrations of NAC. We also created in vitro bacterial biofilms, incubated them with NAC or control, and then stained with propidium iodide and FITC-labeled concanavalin A. We measured biofilm thickness, number of non-viable cells, and fluorescent intensity as a marker of extracellular matrix via a confocal laser scanning microscope. All experiments were conducted in triplicate. Tested organisms included methicillin-sensitive and -resistant Staphylococcus aureus (MSSA, MRSA), S. epidermidis, vancomycin-resistant Enterococcus faecalis (VRE), Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae, Candida albicans and C. krusei. NAC 80 mg/ml was uniformly bactericidal (>99.9% reduction) against all tested bacteria with no recoverable organisms after 30 minutes of incubation, but was fungistatic against candida species. Minimum inhibitory and bactericidal concentrations of NAC ranged from 5-10 mg/ml. Biofilm thickness was significantly decreased in NAC-treated biofilms for all organisms except VRE. The number of non-viable cells in NAC-treated Gram-positive biofilms was increased (p<0.05 for MRSA and VRE). NAC-treated Gram-negative biofilms had scant cellularity and lacked complex 3-dimensional structures that were characteristic of controls. Fluorescent intensity was similar in the experimental and control arms. NAC is bactericidal against clinically relevant and drug-resistant bacteria and also leads to biofilm disruption. NAC has the potential for use as a novel agent for prevention or treatment of biofilm-related infections.

The in-vitro and in-vivo inhibitory activity of biflorin in melanoma

Biflorin, an ortho-naphthoquinone, is an active compound found in the roots of Capraria biflora L. It has been reported that biflorin presents anticancer activity, inhibiting both tumor cell line growth in culture and tumor development in mice. The aim of this study was to examine the effectiveness of biflorin treatment using both in-vitro and in-vivo melanoma models. Biflorin displayed considerable cytotoxicity against all tested cell lines, with half maximal inhibitory concentration values ranging from 0.58 μg/ml in NCI H23 (human lung adenocarcinoma) to 14.61 μg/ml in MDA-MB-231 (human breast cancer) cell lines. In a second set of experiments using B16 melanoma cells as a model, biflorin reduced cell viability but did not cause significant increase in the number of nonviable cells. In addition, the DNA synthesis was significantly inhibited. Flow cytometry analysis showed that biflorin may lead to an apoptotic death in melanoma cells, inducing DNA fragmentation and mitochondria depolarization, without affecting membrane integrity. In B16 melanoma-bearing mice, administration of biflorin (25mg/day) for 10 days inhibited tumor growth, and also increased the mean survival rate from 33.3±0.9 days (control) to 44.5±3.4 days (treated). Our findings suggest that biflorin may be considered as a promising lead compound for designing new drugs to be used in the treatment of melanoma.

Validation of cell density and viability assays using Cedex automated cell counter

A method using Cedex automatic cell counter (Innovatis) to determine the cell density and viability of a whole cell-based immunotherapy product has been developed and validated for the assay performance characteristics including specificity, accuracy, precision, linearity, range, and robustness. Instrument-to-instrument variation due to intrinsic differences in handmade flow cells was also evaluated. For cell density, Cedex demonstrated acceptable specificity, accuracy and precision for cell densities ranging from 3.13 × 10 5 to approximately 1.0 × 10 7 cells/mL, with intermediate precision of about 5% relative standard deviation (RSD). However, a marked difference was observed between the two instruments studied and they therefore could not be used interchangeably without additional calibration procedures that went beyond the manufacturer's recommendation. For viability, mixing known numbers of non-viable cells with highly viable cells allowed evaluation of the specificity, accuracy and linearity of the viability determination. Acceptable levels of accuracy (95.3–106.4% recovery) and precision (RSD < 5%) were demonstrated for the viability range from 50 to 100%. The instrument-to-instrument difference was less than 4.6%. The assays for both cell density and viability were sufficiently robust for assay parameters. However, the effect of certain parameters was cell line-dependent, suggesting that Cedex performance should be verified for each cell line of interest.

Human chorionic gonadotropin (hCG) interacts with lovastatin and ionizing radiation to modulate prostate cancer cell viability in vivo

The ability of human chorionic gonadotropin (hCG) to modify prostate carcinoma viability in vitro and in vivo when combined with the HMG CoA reductase inhibitor lovastatin and ionizing radiation was investigated. Treatment of PC-3 cells in vitro with hCG caused a modest increase in numbers of non-viable cells within 96h. Treatment of cells with hCG followed by exposure to the HMG CoA reductase inhibitor lovastatin suppressed AKT phosphorylation and enhanced the cytotoxic effects of hCG. The cytotoxic effects of hCG were blocked by expression of BCL-XL and dominant negative caspase 9. Treatment of mice bearing PC-3 flank tumors with lovastatin and hCG significantly reduced tumor volume within 7 days; this was also reflected in decreased ex vivo colony survival of the cells which correlated with increased cleavage of pro-caspase 3 and reduced Ki67 immuno-reactivity. In vitro, treatment of PC-3 cells with hCG followed by exposure to ionizing radiation enhanced the cytotoxic effects of hCG, that was further enhanced by lovastatin. In vivo, hCG radiosensitized PC-3 tumors and significantly enhanced the lethality of hCG and lovastatin treatment. Collectively, our findings argue that treatment of PC-3 prostate cancer tumors with hCG, lovastatin and radiation represents a potential novel therapeutic approach.

Thermal sensitization through ROS modulation: A strategy to improve the efficacy of hyperthermic intraperitoneal chemotherapy

The purpose of this study was to investigate whether modulation of cellular reactive oxygen species (ROS) provides a synergistic effect with hyperthermia to induce tumor cell death in a colon cancer cell line. HT-29 colon cancer cells were exposed to heat (43 degrees C) in the presence of the ROS-generating drug, 2-2'-azobis-(2-amidinopropane) dihydrochloride (AAPH) for 1 h. Viable cell mass and apoptosis was measured by MTT and annexin V staining, respectively. Oxidative stress was evaluated by DCFH fluorescence. Protein levels were determined by Western blot analysis. A synergistic effect on cell viability with AAPH was noted under hyperthermic conditions as compared with hyperthermia alone (P < .05). The number of nonviable cells after hyperthermia and AAPH exposure was also significantly increased compared with AAPH at 37 degrees C (42% vs 20%, P < .05). ROS levels were increased modestly with AAPH at 37 degrees C, whereas they increased significantly in a dose-dependent manner with AAPH at 43 degrees C. Transient increases of phosphorylated-p38 and ERK and decreases in phosphorylated-AKT were observed in the cells exposed to AAPH at 43 degrees C. Pretreatment of inhibitors of p38 yielded additional decreases in cell mass when used in combination with AAPH and hyperthermia (P < .05). Increased expression of HSP 27 observed at 43 degrees C was abrogated with AAPH exposure. Oxidative stress increased the cytotoxic effects of hyperthermia in colon cancer cells. Thermal sensitization through modulation of cellular ROS may represent a novel approach to increase the efficacy of hyperthermia as an anticancer modality.

In vitro cytotoxicity against different human cancer cell lines of laticifer proteins of Calotropis procera (Ait.) R. Br

This work evaluated the in vitro cytotoxic activity of laticifer proteins (LP) recovered from the latex of the medicinal plant Calotropis procera. The LP displayed considerable cytotoxicity with IC 50 values ranging from 0.42 to 1.36 μg/ml to SF295 and MDA-MB-435 cell lines, respectively. In healthy peripheral blood mononuclear cells exposed to LP (10 μg/ml) for 72 h, no noticeable effects on viability or cell morphology were seen. The fractionating of LP on an ion exchange chromatography gave rise to a new fraction (PI) that retained almost all cytotoxicity. The cytotoxic effects of both LP and PI were diminished when previously treated with pronase, or 2-mercaptoethanol, suggesting a protein nature of active molecules, however, pre-incubation with dithiothreitol (DTT) only reduced PI activity. PI did not exhibit cysteine proteinase activity, indicating that cysteine proteinases, abundantly found in LP, are not implicated in LP cytotoxicity. In this study, using HL-60 cell as a model, LP was shown to inhibit DNA synthesis. This is probably due to alterations in the topology of DNA, since it was observed that LP is able to interfere in topoisomerase I activity by somehow acting upon DNA. LP provoked reduction in cell number but it did not cause any significant increase in the number of non-viable cells. These findings corroborated with the morphologic analysis, where cells treated with LP showed morphology of apoptotic process with abundant vacuoles, chromatin condensation and fragmentation of the nuclei. The results of this study suggests that LP is a target for DNA topoisomerase I triggering apoptosis in cancer cell lines.

Regulation of apoptosis in the atresia of dominant bovine follicles of the first follicular wave following ovulation

During atresia of bovine follicles, granulosa cells are lost through the controlled form of cell death, apoptosis. The purpose of this study was to characterize the regulation of apoptotic death of granulosa cells in dominant bovine follicles during the first wave of follicular development. Dominant follicles were collected from Holstein heifers on days 4, 6 or 8 of the first follicular wave (n = 5/day). Regulation of apoptosis in granulosa cells was examined by annexin V and propidium iodide staining; measurement of relative levels of mRNA encoding Bcl-2, Bcl-xL and Bax; and activity of caspase-3, -8 and -9. Steady-state levels of mRNA encoding four oxidative stress-response proteins were determined. Compared with day 4, the incidence of apoptotic and nonviable granulosa cells tended to increase on day 6, and numbers of nonviable cells were higher on day 8. The ratios of relative levels of mRNA encoding Bcl-2 to Bax and Bcl-xL to Bax were higher on day 6 than days 4 and 8. Activity of caspases-3 and -9 in granulosa cells did not change among the 3 days, while caspase-8 activity decreased on day 8 compared with days 4 and 6. Amounts of GSHPx, MnSOD and Cu/ZnSOD mRNA in granulosa cells were higher on day 8 than day 6. In theca interna, amounts of Cu/ZnSOD mRNA decreased between days 4 and 6. From the decreased production of estradiol and increased numbers of apoptotic and nonviable granulosa cells, we conclude that atresia of the dominant follicle is initiated between days 4 and 6 of the first follicular wave. However, apoptosis of granulosa cells does not appear to be initiated by changes in expression of oxidative stress-response proteins.

Early protective effects of iloprost, a stable prostacyclin analog, during spinal cord ischemia in a rabbit model

Spinal cord ischemia may develop into paraplegia in some cases during operation of the thoracoabdominal aorta. This is attributable to the vulnerability of spinal motor neurons to ischemia. In this study, iloprost was used as an agent to decrease the severity of ischemia and reperfusion injury to the spinal cord motor neurons. Twenty-one rabbits were randomized into three groups of seven animals each: group A (iloprost not administered), group B (25 ng/kg per minute iloprost), and group S (sham-operated). The spinal cord ischemia model was created by a 15-min occlusion of the aorta just caudal to the renal artery with a balloon catheter. Administration of iloprost began 10 min before occlusion of the aorta, and continued thereafter for 60 min. The pre- and postocclusion arterial pressure and heart rate recordings, results of blood gas analyses, and hematocrit and glucose levels were recorded. The spinal cords were removed after 8-h monitoring of neurologic function. Viable and nonviable motor neurons in the anterior horn of the spinal cord were counted under light microscopy. Any significant alteration in hemodynamics, blood gases, and other physiologic parameters could not be detected within the groups. Iloprost had a moderately hypotensive effect. Neurologic function in terms of Johnson scoring was significantly better in the iloprost group (P<0.05). The number of viable cells was higher, whereas the number of nonviable cells was lower in iloprost group, when compared with the control group (P<0.05). Higher numbers of viable motor neurons were consistent with the neurological findings. As a result of this study we concluded that iloprost infused during clamping of the aorta mitigates the spinal cord injury due to ischemia and reperfusion, and has a significant protective effect.

A novel porcine dry eye model system (pDEM) with simulated lacrimation/blinking system: Preliminary findings on system variability and effect of corneal drying

Purpose. To investigate inter- and intra-system variations and the effect of corneal drying using a recently developed pDEM. Methods. pDEM was used to simulate “normal” and “dry eye” conditions using two “lacrimation-blink” intervals (20 s and 60 s). Corneas were examined/graded with sodium fluorescein before and after the experiment. At the end of each experiment, corneas were assessed by trypan blue exclusion technique. Two duplicated pDEM systems were set up and tested to investigate reproducibility. Results. There was no significant difference in the results produced by the two pDEM systems. In the eyes under “normal” condition, there was no significant increase in the fluorescein grading. However, in eyes under “dry eye” condition, fluorescein staining increased and the number of non-viable cells in the central cornea increased. Conclusions. This novel pDEM system provides a useful assessment tool for the study of causative factors and new treatment strategies for dry eye syndrome.

Effect of eyelid closure and overnight contact lens wear on viability of surface epithelial cells in rabbit cornea.

To determine the effects of open, closed eye, and overnight contact lens wear on homeostatic epithelial surface cell death in the rabbit cornea. One eye of each rabbit was either closed by eyelid suture or fitted with one of the following test contact lenses: (1) low Dk/t rigid gas permeable (RGP) lens, (2) hyper Dk/t RGP lens, (3) hyper Dk/t soft lens. The other eye served as a control. After 24 hours, whole corneas were carefully excised and immediately stained with a calcein-acetoxymethyl ester-ethidium homodimer viability assay to quantify the number of nonviable surface epithelial cells. In addition, exfoliated corneal epithelial cells were collected with an eye irrigation chamber to determine cell viability. In the normal cornea, open-eye conditions showed significantly more nonviable surface cells in the central cornea than in the periphery (p < 0.05). Overnight wear of all test lenses and eyelid closure induced significant decreases in the number of nonviable cells on the central corneal surface compared with controls (p < 0.05). All exfoliated corneal epithelial cells collected by eye irrigation were nonviable. In the rabbit model, overnight contact lens wear significantly downregulated spontaneous epithelial surface cell death independent of lens rigidity or material oxygen transmissibility. These effects were similar to eyelid closure without lens wear. Taken together, these results suggest that eyelid closure and the physical presence of the contact lens may protect against the shear stress forces exerted by eyelid blinking, which are believed to cause central surface cell death and subsequent exfoliation.

Skeletal muscle microvascular and tissue injury after varying durations of ischemia.

The purpose of this study was to examine the effect of varying durations of ischemia on several microvascular parameters in the awake hamster chamber model. The goal was to characterize the microvascular damage that occurs in skeletal muscle as a result of ischemia and reperfusion. The chamber tissues were subjected to 1-5 h of ischemia, and then the following parameters were measured: vessel diameter, endothelial thickness, macromolecular leakage, red blood cell velocity, adherent leukocytes, rolling leukocytes, freely flowing leukocytes, functional capillary density, and propidium iodide-positive cell nuclei. In control animals there was no significant difference in any parameters over the entire observation period. After 1 or 2 h of ischemia an increase in rolling and adherent leukocytes was measured. After 3 h of ischemia there was a significant increase in the mean endothelial thickness and in the number of nonviable cells. After 4 h of ischemia a significant difference in the extent of macromolecular leakage and the functional capillary density was additionally observed. After 5 h of ischemia this damage was more pronounced and often so severe that approximately 50% of the vessels demonstrated no reflow.